PROJECT SUMMARY Type 2 diabetes (T2D) is caused by a failure of beta cells to produce sufficient insulin to maintain euglycemia. As a consequence of genetic/environmental factors, insulin resistance develops that pressures beta cells to increase insulin production. Although beta cells have some capacity to compensate for the demand, by approximately one-third of ~600 million individuals with obesity in the world develop go on to develop diabetes. The factors that lead to beta cell failure are unknown. Due to the polygenic nature of the disease, it is likely many genes modify beta cell function, and polymorphisms in any single gene would not be detected because they present minor contributions. Our underlying hypothesis is that multiple genes impact the efficiency of proinsulin folding in the endoplasmic reticulum (ER) and modify the progression of T2D. Significantly, our preliminary studies show that a high fat diet is sufficient to cause proinsulin misfolding well before diabetes development in C57BL/6 mice. In addition, we have identified genetic modifiers that exacerbate proinsulin misfolding and beta cell failure. We hypothesize that the fundamental cause of beta cell failure in T2D is a breakdown at the level of the ER with failure to efficiently fold excessive amounts of proinsulin and resulting consequences on downstream processing and secretion. To test our hypothesis, we have established a team of outstanding investigators to work together to identify critical proteins that modify proinsulin folding using state-of-the-art proteomics, biochemistry, cell biology, murine genetics and bioinformatics. In preliminary studies we developed methods to differentiate between specific disulfide bond defects and other misfolded conformations of proinsulin, generated all of the necessary murine strains and validated the proteomic mass spectrometry approach for proinsulin interactions using human islets. We have also demonstrated the potential of small molecules to improve proinsulin production in challenged islets. We expect our novel approach will identify distinct defects in the proinsulin folding pathway that represent the earliest changes leading to beta cell demise in both murine models and humans. The three aims of our R24 grant focus on defining how proinsulin folding patterns change when islets are challenged, and to identify how protein interactions with proinsulin may predict the efficiency of proinsulin trafficking through the secretory pathway, impacting islet health. Aim 1 will quantify the folded and unfolded state of proinsulin by measuring intermediates in the folding process in normal and diseased islets from well-characterized murine models. Aim 2 will define how the proteins that interact with proinsulin change during progression from normal, obese non-diabetic to T2D islets from human donors. Aim 3 will elucidate what interventions and chaperone functions may preserve productive proinsulin folding and restore an efficient proinsulin ?proteostasis? network. Collectively, our proposed studies may identify novel biomarkers and avenues for therapeutic intervention in T2D, and therefore are of paramount importance to the mission of NIDDK.